Abstract Chronic toxicity studies are essential for evaluating potential adverse effects associated with the long-term or repeated administration of pharmaceutical compounds, biologically active substances, herbal products, and emerging materials. These studies aim to identify cumulative and organ-specific toxicities that may not be detected in short-term assessments, thereby supporting dose selection, risk characterization, and regulatory decision-making. This review summarizes current methodologies, key endpoints, regulatory considerations, and recent scientific advances in chronic toxicity evaluation using laboratory animal models.
The review is based on the Organisation for Economic Co-operation and Development (OECD) Test Guidelines 407, 408, and 452, which define standardized approaches for 28-day, 90-day, and long term repeated-dose toxicity studies in rodents. Core evaluation parameters discussed include clinical observations, body weight and food consumption, hematological and biochemical analyses, urinalysis, endocrine assessments, organ weights, necropsy findings, and histopathological examinations, with emphasis on the determination of the no observed adverse effect level (NOAEL).
In addition to conventional endpoints, recent developments such as ophthalmological examinations, molecular and oмics-based technologies, and novel biomarkers are highlighted for their ability to improve mechanistic understanding and early detection of organ injury. The integration of alternative in vitro models, organoids, organ-on-a-chip systems, and in silico approaches in accordance with the 4R principles is also discussed. Despite existing limitations, chronic toxicity studies remain a cornerstone of preclinical safety assessment, and their integration with advanced technologies enhances relevance, ethical compliance, and predictive value.